Endothelial DR6 in blood-brain barrier malfunction in Alzheimer's disease.

The impairment of the blood-brain barrier (BBB) has been increasingly recognised as a critical element in the early pathogenesis of Alzheimer's disease (AD), prompting a focus on brain endothelial cells (BECs), which serve as the primary constituents of the BBB. Death receptor 6 (DR6) is highly expressed in brain vasculature and acts downstream of the Wnt/ß-catenin pathway to promote BBB formation during development. Here, we found that brain endothelial DR6 levels were significantly reduced in a murine model of AD (APPswe/PS1dE9 mice) at the onset of amyloid-ß (Aß) accumulation. Toxic Aß25-35 oligomer treatment recapitulated the reduced DR6 in cultured BECs. We further showed that suppressing DR6 resulted in BBB malfunction in the presence of Aß25-35 oligomers. In contrast, overexpressing DR6 increased the level of BBB functional proteins through the activation of the Wnt/ß-catenin and JNK pathways. More importantly, DR6 overexpression in BECs was sufficient to rescue BBB dysfunction in vitro. In conclusion, our findings provide new insight into the role of endothelial DR6 in AD pathogenesis, highlighting its potential as a therapeutic target to tackle BBB dysfunction in early-stage AD progression.

Hugan Qingzhi tablets attenuates endoplasmic reticulum stress in nonalcoholic fatty liver disease rats by regulating PERK and ATF6 pathways.

BACKGROUND: Endoplasmic reticulum (ER) stress, promoting lipid metabolism disorders and steatohepatitis, contributes significantly to the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Hugan Qingzhi tablets (HQT) has a definite effect in the clinical treatment of NAFLD patients, but its mechanism is still unclear. This study aims to investigate the effects of HQT on ER stress in the liver tissues of NAFLD rats and explore the underlying mechanism. METHODS: The NAFLD rat model was managed with high-fat diet (HFD) for 12weeks. HQT was administrated in a daily basis to the HFD groups. Biochemical markers, pro-inflammatory cytokines, liver histology were assayed to evaluate HQT effects in HFD-induced NAFLD rats. Furthermore, the expression of ER stress-related signal molecules including glucose regulating protein 78 (GRP78), protein kinase RNA-like endoplasmic reticulum kinase (PERK), p-PERK, eukaryotic translation initiation factor 2α (EIF2α), p-EIF2α, activating transcription factor 4 (ATF4), acetyl-coenzyme A-carboxylase (ACC), activating transcription factor (ATF6), and nuclear factor-kappa B-p65 (NF-κB-p65) were detected by western blot and/or qRT-PCR. RESULTS: The histopathological characteristics and biochemical data indicated that HQT exhibited protective effects on HFD-induced NAFLD rats. Furthermore, it caused significant reduction in the expression of ERS markers, such as GRP78, PERK, p-PERK, and ATF6, and subsequently downregulated the expression of EIF2α, p-EIF2α ATF4, ACC, and NF-κB-p65. CONCLUSIONS: The results suggested that HQT has protective effect against hepatic steatosis and inflammation in NAFLD rats by attenuating ER stress, and the potential mechanism is through inhibition of PERK and ATF6 pathways.

Berberine alleviates inflammation and suppresses PLA2-COX-2-PGE2-EP2 pathway through targeting gut microbiota in DSS-induced ulcerative colitis.

Berberine, isolated from Coptis chinensis and Phellodendron amurense, can attenuate colonic injury and modulate gut microbiota disorders in ulcerative colitis (UC). However, the mechanism and causal relationship between gut microbiota and the efficacy of Berberine on UC are still unclear, which were investigated by pseudo-germ-free (PGF) mice, 16S rRNA gene analysis and transcriptome analysis in this study. The results demonstrated that Berberine improved gut microbiota disorders, colon damage, tight-junction proteins, inflammatory and anti-inflammatory cytokines in DSS-induced colitis mice with intact gut microbiota but not in PGF mice. Besides, immune-related and inflammation-related pathways were closely related to the efficacy that Berberine alleviated colitis by regulating gut microbiota. Furthermore, Berberine reduced PGE2, PLA2, COX-2, Ptges, EP2 and p-Stat3 only in colitis mice with intact gut microbiota. In summary, our study confirms that Berberine inhibits PLA2-COX-2-PGE2-EP2 pathway in UC through gut microbiota, leading to the alleviation of inflammation in colon, which further elucidates the underlying mechanism and promotes the application of Berberine in UC.

Dual-Targeting Nanovesicles Carrying CSF1/CD47 Identified from Single-Cell Transcriptomics of Innate Immune Cells in Heart Transplant for Alleviating Acute Rejection.

Although immunosuppressive drugs for targeting T cells are the standard of care in acute transplantation rejection, the role of innate immune cells should not be ignored. Here, single-cell RNA sequencing (scRNA-seq) and flow cytometry are performed to reveal the dynamic changes of innate immune cells within the acute rejection time and find a significantly-increased presence of Ly6G- Ly6C+ inflammatory macrophages and decreased presence of neutrophils among all types of immune cells. Next, to further explore potential targets regulating Ly6G- Ly6C+ inflammatory macrophages, scRNA-seq is used to analyze the reciprocal signaling of both neutrophils and macrophages, along with the surface genes of macrophages. It is found that activating colony-stimulating factor 1/ colony-stimulating factor 1 receptor (CSF1/CSF1R) andcluster of differentiation 47/signal regulatory protein α (CD47/SIRPα) signaling may serve as a strategy to relieve Ly6G- Ly6C+ inflammatory macrophage-mediated early graft rejection. To investigate this hypothesis, CSF1/CD47 dual-targeting nanovesicles (NVs) derived from IFN-γ-stimulated induced pluripotent stem cell-derived mesenchymal stem cells ( iPSC-MSCs )are designed and constructed. It is confirmed that CSF1/CD47 NVs synergistically induce the differentiation of Ly6G- Ly6C- M2 inhibitory macrophages by the CSF1/CSF1R pathway, and inhibit the phagocytosis of inflammatory macrophages and inflammatory response by the CD47/SIRPα pathway, ultimately relieving immune rejection. This study highlights the power of dual-targeting CSF1/CD47 NVs as an immunosuppressant against early innate immune responses with the potential for broad clinical applications.

Isoliquiritigenin suppresses the progression of malignant melanoma via targeting H2A.Z.1-E2F1 pathway.

Cutaneous melanoma is one of the most prevalent tumors, and it is still a huge challenge in the current clinical treatment. Isoliquiritigenin (ISL), which is isolated from Glycyrrhiza uralensis Fisch., has been reported for its anti-tumor effect. However, the underlying mechanism and targets of ISL are still not be revealed clearly. In this study, differentiallyexpressedproteins were identified bylabel-free quantitative mass spectrometry. Two isoforms of the histone variant H2A.Z, including H2A.Z.1 and H2A.Z.2, were significantly down regulated after administration of ISL in melanoma. H2A.Z.1 was highly expressed in melanoma and correlated with poor prognosis of melanoma. The expression of H2A.Z was inhibited by ISL in a concentration-dependent manner. Overexpression of H2A.Z.1 in melanoma cell lines partly restored the repressed cell proliferation and cell cycle by ISL. Moreover, E2F1 was identified as one downstream target of H2A.Z.1, which was also highly expressed in melanoma and correlated with poor prognosis of melanoma. Furthermore, in vivo assays validated the inhibitory role of ISL in melanoma proliferation and the expression of H2A.Z.1 and E2F1.Aboveall,it is indicated that ISL inhibit melanoma proliferation via targeting H2A.Z.1-E2F1 pathway. These findings explain the anti-tumor mechanism of ISL and provide potential therapeutic targets for melanoma.

Alisol B 23-Acetate Ameliorates Lipopolysaccharide-Induced Intestinal Barrier Dysfunction by Inhibiting TLR4-NOX1/ROS Signaling Pathway in Caco-2 Cells.

Alisol B 23-Acetate (AB23A) is a naturally occurring triterpenoid, which can be indicated in the rhizome of medicinal and dietary plants from Alisma species. Previous studies have demonstrated that AB23A could inhibit intestinal permeability by regulating tight junction (TJ)-related proteins. Even so, the AB23A protective mechanism against intestinal barrier dysfunction remains poorly understood. This investigation seeks to evaluate the AB23A protective effects on intestinal barrier dysfunction and determine the mechanisms for restoring intestinal barrier dysfunction in LPS-stimulated Caco-2 monolayers. According to our findings, AB23A attenuated the inflammation by reducing pro-inflammatory cytokines production like IL-6, TNF-α, IL-1ß, and prevented the paracellular permeability by inhibiting the disruption of TJ in LPS-induced Caco-2 monolayers after treated with LPS. AB23A also inhibited LPS-induced TLR4, NOX1 overexpression and subsequent ROS generation in Caco-2 monolayers. Transfected with NOX1-specific shRNA diminished the up-regulating AB23A effect on ZO-1 and occludin expression. Moreover, transfected with shRNA of TLR4 not only enhanced ZO-1 and occludin expression but attenuated NOX1 expression and ROS generation. Therefore, AB23A ameliorates LPS-induced intestinal barrier dysfunction by inhibiting TLR4-NOX1/ROS signaling pathway in Caco-2 monolayers, suggesting that AB23A may have positive impact on maintaining the intestinal barrier's integrity.

Ficus hirta Vahl. Ameliorates Nonalcoholic Fatty Liver Disease through Regulating Lipid Metabolism and Gut Microbiota.

Nonalcoholic fatty liver disease (NAFLD) has gradually become one of the most serious liver diseases threatening human health in the world. Currently, Chinese herbal medicine is a potentially important treatment option for NAFLD, and the development of effective Chinese herbal medicine has a good prospect. Previous studies have suggested that Ficus hirta Vahl. (FV) has various protective effects on the liver. In this study, we investigated the therapeutic outcomes of FV treatment for the liver disease and its underlying mechanism using HepG2 cell lines induced by palmitate (PA) and mouse model fed with high-fat diet (HFD). FV mainly exerts pharmacological effects by mediating lipid metabolism and inflammation. During the lipid metabolism regulation process, CD36, SREBP-1, SCD1, PPAR γ, ACOX1, and CPT1α are the key factors related to the healing effects of FV on NAFLD. During the inflammation process, the downregulation of IL-6, IL-1ß, and TNF-α is involved in alleviation of NAFLD. Furthermore, CD36 overexpression promotes lipid abnormal metabolism and inflammation in PA-induced HepG2 cells, while CD36 knockdown and FV supplementation reverse these responses. In addition, FV also modulates gut microbiota composition, such as Allobaculum, Faecalibaculum, and Butyricicoccus in HFD-fed mice. In summary, our findings demonstrated that FV exerted a beneficial preventive and therapeutic effect on NAFLD by improving lipid metabolism and inflammation as well as regulating the structure of gut microbiota, and therefore, FV may be a candidate for the treatment of NAFLD.

No association between recent antibiotic use and risk of rheumatoid arthritis: results from two prospective cohort studies.

BACKGROUND: Retrospective research partly characterizes the link between antibiotic use and rheumatoid arthritis (RA) development. This prospective cohort study may help reassess the association. RESEARCH DESIGN AND METHODS: We included 133,125 participants from the Nurses' Health Study (NHS) and NHS II databases. Three groups were established: nonuse, short-term use (1-14 days), and middle- to long-term use (≥15 days) to explore the link. Cox regression model was chosen to evaluate the hazard ratios (HRs) for RA. RESULTS: Short-term antibiotic use was not associated with the subsequent risk of RA (adjusted HR = 0.88, 95% Confidence Interval [CI] 0.38-1.38) compared to the no antibiotic use group in the multivariable adjusted model. The age-stratified model showed no sufficient evidence of increased risk in participants with middle- to long-term antibiotic use (HR = 1.32, 95% CI 0.89-1.98). The effect further attenuated to null after controlling for confounding factors (adjusted HR = 1.06, 95% CI 0.42-1.71). CONCLUSIONS: We found no evidence of an association between antibiotic use and RA risk. Our findings may reduce potential concerns about increased RA risk among antibiotic users.

The probiotic effects of AB23A on high-fat-diet-induced non-alcoholic fatty liver disease in mice may be associated with suppressing the serum levels of lipopolysaccharides and branched-chain amino acids.

Alisol B 23-acetate (AB23A) is a natural triterpenoid isolated from Rhizoma alisamatis that has been widely used as a traditional Chinese medicine (TCM). Previous studies have documented the beneficial effect of AB23A on non-alcoholic fatty liver disease (NAFLD), but the functional interactions between gut microbiota and the anti-NAFLD effect of AB23A remain unclear. In this study, we investigated the benefits of experimental treatment with AB23A on gut microbiota dysbiosis in NAFLD with an obesity model. C57BL/6J mice were administrated a high-fat diet (HFD) with or without AB23A for 12 weeks. AB23A significantly improved metabolic phenotype in the HFD-fed mice. Moreover, results of 16S rRNA gene-based amplicon sequencing in each group reveled that AB23A not only reduced the abundance of the Firmicutes/Bacteroidaeota ratio and Actinobacteriota/Bacteroidaeota ratio, but regulated the abundance of the top 10 genera, including norank_f__Muribaculaceae, Lactobacillus, Ileibacterium, Turicibacter, Faecalibaculum, the Lachnospiraceae_NK4A136_group, unclassified_f__Lachnospiraceae, and norank_f__Lachnospiraceae. AB23A significantly reduced the serum levels of lipopolysaccharide and branched-chain amino acids, which are positively correlated with the abundances of Ileibacterium and Turicibacter. Moreover, AB23A led to remarkable reductions in the activation of TLR4, NF-κB, and mTOR, and upregulated the expression of tight junction proteins, including ZO-1 and occludin. These results revealed that AB23A displayed a prebiotic capacity in HFD-fed NAFLD mice.

Gut microbiota as a target for prevention and treatment of type 2 diabetes: Mechanisms and dietary natural products.

Type 2 diabetes mellitus (T2DM) is among the most remarkable public health concerns globally. Accumulating research evidence documents that alteration of gut microbiota has an indispensable role in the onset and progression of obesity and T2DM. A reduced microbial diversity is linked to insulin resistance and energy metabolism, especially for the rise of the Firmicutes/Bacteroidetes ratio. Changes in metabolites followed by the gut dysbacteriosis are linked to the presence of T2DM. Moreover, endotoxin leakage and gut permeability caused by gut dysbacteriosis is more of a trigger for the onset and progression of T2DM. Research documents that natural products are remarkable arsenals of bioactive agents for the discovery of anti-T2DM drugs. Many studies have elucidated that the possible mechanisms of the anti-T2DM effects of natural products are remarkably linked to its regulation on the composition of gut microflora and the successive changes in metabolites directly or indirectly. This review presents a brief overview of the gut microbiota in T2DM and several relevant mechanisms, including short-chain fatty acids, biosynthesis and metabolism of branched-chain fatty acids, trimethylamine N-oxide, bile acid signaling, endotoxin leakage, and gut permeability, and describes how dietary natural products can improve T2DM via the gut microbiota.

The dietary flavonoid isoliquiritigenin induced apoptosis and suppressed metastasis in melanoma cells: An in vitro and in vivo study.

AIMS: This study aimed to explore the role of Isoliquiritigenin (ISL) in the proliferation and invasion of melanoma cells and investigate the mechanism of action of this compound. MAIN METHODS: The functional roles of ISL in melanoma cells were determined by CCK8 assay, colony formation assay, flow cytometry and wound healing assay. The antitumor activity of ISL was assessed in vivo in a mouse xenograft model using A2058 cells. Quantitative real-time PCR analysis (RT-qPCR) and western blot assays were used to evaluate the gene and protein expression in cell lines or tumor tissue samples. Bioinformatic analysis, luciferase reporter assay, and gene set enrichment analysis (GSEA) were performed to confirm the mechanism of ISL effect on cell growth and metastasis of melanoma. KEY FINDINGS: ISL suppressed proliferation and migration of melanoma cells via downregulation of miR-27a expression. The inhibitory effect of ISL on growth and metastasis of melanoma cells was reversed by ectopic expression of miR-27a. Bioinformatic analysis showed that miR-27a targets POU class 2 homeobox 3 (POU2F3); this result was verified by the luciferase reporter assay and by a decrease in the expression of POU2F3 by miR-27a intervention. GSEA demonstrated that POU2F3 is associated with the c-MYC/p53 signaling pathway and metastasis. POU2F3 knockdown reversed the inhibitory effect of ISL on the growth and metastasis of melanoma. Additionally, POU2F3 was found to be downregulated in melanoma tissue samples and was negatively correlated with miR-27a. SIGNIFICANCE: ISL inhibits proliferation and metastasis of melanoma via the miR-27a/POU2F3/c-MYC/p53 axis; these results may provide a new thought for the treatment of melanoma.

Hugan Qingzhi medication ameliorates free fatty acid-induced L02 hepatocyte endoplasmic reticulum stress by regulating the activation of PKC-δ.

BACKGROUND: Previous studies have found that Hugan Qingzhi tablet (HQT) has significant lipid-lowering and antioxidant effects on non-alcoholic fatty liver disease (NAFLD). Moreover, the results of proteomic analysis confirmed that various proteins in endoplasmic reticulum stress (ERS) pathway were activated and recovered by HQT. However, its mechanism remains confused. The purpose of this study was to explore the effects of HQT-medicated serum on hepatic ERS and its relevant mechanisms. METHODS: L02 cells were induced by Free Fatty Acid (FFA) for 24 h to establish a model of hepatic ERS and pretreated with the drug-medicated rat serum for 24 h. Accumulation of intracellular lipid was evaluated using Oil Red O staining and Triglyceride detection kit. The morphological changes of ER were observed by TEM. PKC-δ was silenced by specific siRNA. Western blot and RT-qPCR were applied to detect the expression of markers related to ERS, calcium disorder, steatosis and insulin resistance. The fluorescence of Ca2+ influx was recorded using fluorescence spectrophotometer. RESULTS: HQT-medicated serum significantly decreased the intracellular TG content. Furthermore, it caused significant reduction in the expression of ERS markers and an improvement in ER structure of L02 cells. PKC-δ was activated into phosphorylated PKC-δ in FFA-induced L02 hepatocytes while these changes can be reversed by HQT-medicated serum. Silencing PKC-δ in L02 cells can restore the expression and activity of SERCA2 in ER and down-regulate the expression of IP3R protein to maintain intracellular calcium homeostasis, so as to relieve FFA-induced ERS and its lipid accumulation and insulin resistance. CONCLUSIONS: The results concluded that HQT-medicated serum exerts protective effects against hepatic ERS, steatosis and insulin resistance in FFA-induced L02 hepatocyte. And its potential mechanism might be down-regulating the activation of PKC-δ and stabilization of intracellular calcium.

Sodium aescinate significantly suppress postoperative peritoneal adhesion by inhibiting the RhoA/ROCK signaling pathway.

BACKGROUND: Although mechanical barriers and modern surgical techniques have been developed to prevent postoperative adhesion formation, high incidence of adhesions still represents an important challenge in abdominal surgery. So far, there has been no available therapeutic drug in clinical practice. PURPOSE: In this study, we explored the efficacy of sodium aescinate (AESS) treatment against postoperative peritoneal adhesions, the potential molecular mechanism was also investigated. STUDY DESIGN AND METHODS: Sixty male Sprague-Dawley rats were randomly divided into 6 groups for the study: the blank, vehicle, positive control and three AESS administration groups (0.5, 1 and 2 mg/kg/d, intravenous administration for 7 days). Adhesions were induced by discretely ligating peritoneal sidewall. An IL-1ß-induced HMrSV5 cell model was also performed to explore possible functional mechanism. RESULTS: The results indicated that the incidence and severity of peritoneal adhesions were significantly lower in the AESS-treated groups than that in the vehicle and positive control group. AESS-treated groups showed that the secretion, activity, and expression of tPA in rat peritoneum were notably increased. The FIB levels in rat plasma were decreased. The immunohistochemical staining analysis demonstrated that collagen I and α-SMA deposition were significantly attenuated in AESS-treated peritoneal tissues. Besides, we found that AESS treatment reduced the protein levels of p-MYPT1. To further explore the mechanisms of AESS, both activator and inhibitors of RhoA/ROCK pathway were employed in this study. It was found that AESS-induced up-regulation of tPA was reversed by activator of ROCK, but the effects of ROCK inhibitors were consistent with AESS. CONCLUSION: Taken together, the findings of in vivo and in vitro experiments proved that AESS could significantly suppress postoperative peritoneal adhesion formation through inhibiting the RhoA/ROCK signaling pathway. Our researches provide important pharmacological basis for AESS development as a potential therapeutic agent on peritoneal adhesions.

Discovery of Quality Markers in Hugan Qingzhi Formula by Integrating a Lipid-Lowering Bioassay with UHPLC-QQQ-MS/MS.

Nonalcoholic fatty liver disease (NAFLD) is a prevalent chronic liver disease. The Hugan Qingzhi formula (HGQZ) has been proven effective in treating NAFLD through clinical and pharmacological mechanism studies. A screening study of the chemical components was carried out to better control the quality of this formula. Current research has combined biological activity assessment with chemical analysis to screen and identify the bioactive compounds in HGQZ for use as potential quality markers (Q-markers) to control the quality of this herbal product. The HGQZ extracted by three different solvents was evaluated in a free fatty acid-induced hepatic steatosis LO2 cell model. Simultaneously, the twelve major chemical constituents of these extracts were quantitatively measured by ultrahigh-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-QQQ-MS/MS). Extraction with 50% ethanol showed the most potent lipid-lowering effect in steatosis LO2 cells and the highest extraction rate of major chemical constituents. Correlation analysis was used to establish the relationship between the biological activities and chemical characteristics of these extracts. The results showed that the contents of typhaneoside, hyperoside, isoquercitrin, isorhamnetin-3-O-neohesperidoside, notoginsenoside R1, and alisol B 23-acetate were positively correlated to the lipid-lowering effect. The subsequent bioassay confirmed that typhaneoside, isoquercitrin, and alisol B 23-acetate played the role of reducing the lipid effect. In conclusion, 50% of ethanol extraction produced the most active extract of HGQZ. Typhaneoside, isoquercitrin, and alisol B 23-acetate could be considered potential Q-markers for the quality control of HGQZ.

Berberine ameliorates colonic damage accompanied with the modulation of dysfunctional bacteria and functions in ulcerative colitis rats.

Intestinal flora imbalance is one of the potential pathogenesis of inflammatory bowel diseases, and the study aims to discover the effect of berberine on the composition and function of gut microbiota in ulcerative colitis (UC) rats. UC rats were induced by dextran sulfate sodium (DSS) and administrated with berberine. Colonic morphological changes and claudin-1 protein of colon tissues were primarily examined to validate the protective effects brought by berberine treatment. Then the composition and function of gut microbiota were analyzed, accompanied with quantitative analysis of serum amino acids. The results showed that berberine could not only ameliorate the colonic damages in DSS-induced UC rats but also regulate the gut microbiota by increasing lactic acid-producing bacteria and carbohydrate hydrolysis bacteria as well as decreasing conditional pathogenic bacteria. Accordingly, the relevant functions of above bacteria were improved, including the metabolism and biosynthesis of amino acids, capability of DNA replication and repair, carbohydrate digestion and absorption and glycolysis/gluconeogenesis. Furthermore, the serum amino acids were regulated and showed high correlation with the gut microbiota after berberine treatment. In conclusion, the study confirms the effect of berberine on ameliorating the colonic damage and highlights some specific bacteria and relevant functions linked with berberine treatment, exploring the potential of gut microbiota as a diagnostic biomarker or a therapeutic target in UC treatment.

Experimental and simulation research on the screening effect of the pixelated CZT imaging detector.

Massive efforts have been made to investigate the characteristics of the CdZnTe detector under different extreme conditions. In this paper, we experimented with different radiation sources to investigate the imaging screening effect of the pixelated CdZnTe detector under ultrahigh irradiance. A donut-shaped irradiation image was obtained due to the increase in the X-ray tube current or the tube voltage. Moreover, statistical data revealed that the total count of all pixels was not significantly different, while the event-count of irradiated area pixels decreased significantly. A consequence of the screening effect was that the event counts redistributed among pixels that are located in the irradiated area, while the inner electric field was distorted. This leads to the catastrophic performance degradation of the central pixels. According to the Poisson equation, we developed a theoretical model of the CdZnTe detector using the finite element software COMSOL to enable an in-depth investigation of carrier collection in the CdZnTe crystal. A comparison between the simulations and the test results showed that pixels in the central irradiated area are completely screened under the ultrahigh irradiance because of the emergence of the relatively higher potential region, which can distort the electron drift path. Furthermore, the photon-generated signal can be collected only partly at the edge of the irradiated area, which was relatively stable because the collection area in the CdZnTe volume was relatively limited. The imaging results deduced from the simulations are well consistent with the experimental data.

Isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomics for the investigation of the effect of Hugan Qingzhi on non-alcoholic fatty liver disease in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Hugan Qingzhi tablet (HQT), a traditional Chinese medicine formula has been adopted for preventing and treating nonalcoholic fatty liver disease (NAFLD). AIM: In order to explore the anti-NAFLD mechanisms of HQT, iTRAQ-based proteomic was employed to investigate the expression profiles of proteins in NAFLD rats induced by high-fat diet after HQT treatment. MATERIALS AND METHODS: The NAFLD rat model was administrated with high-fat diet (HFD) for 12weeks. HQT was administrated in a daily basis to the HFD groups. Biochemical markers, liver histology, pro-inflammatory cytokines, and oxidative stress/antioxidant biomarkers were assayed to evaluate HQT effects in HFD-induced NAFLD rats. Furthermore, the combined strategy of iTRAQ labeling with strong cation exchange-non-liquid chromatography-tandem mass spectrometry (SCX-non-LC-MS/MS) analysis were employed to explore the mechanisms of HQT's protective effect against NAFLD in rats. Western blotting was performed to verify the proteomic results. RESULTS: The histopathologic characteristics and biochemical data showed that HQT exhibited protective effects on HFD-induced NAFLD rats. After being analyzed by the combined strategy of iTRAQ with LC-MS/MS and subsequent investigation, we identified 275 differentially expressed proteins in the HFD group, compared to the control; 207 altered proteins in the HQT high dose + HFD group, compared to the HFD group; and 316 altered proteins in the HQT high dose + HFD group, compared to the control. Based on the Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway mapping, the conclusion has reached that several pathways including microbial metabolism in diverse environments, fatty acid metabolism, inflammatory response, oxidative stress, bile secretion, and peroxisome proliferator activated receptor (PPAR) signaling pathway were closely related to the effects of HQT in HFD-induced NAFLD in rats. Furthermore, several differentially expressed proteins, including phytanoyl-CoA 2-hydroxylase (PHYH), acyl-CoA synthetase 1 long chain (ACSL1), hemopexin, Alpha-1-acid glycoprotein (ORM1), fatty acid binding protein 4 (FABP4), soluble sulphotransferase 2a1 (Sult2a1), and argininosuccinate synthase 1 (ASS1) were verified by the western blotting analysis and these results were consistent with the data obtained from the proteomics analysis. CONCLUSIONS: Our results not only confirm that Hugan Qingzhi exhibits a significant protective effect in HFD-induced NAFLD rats but also provide a better understanding for the treatments of NAFLD.

Modulation of the Gut Microbiota in Rats by Hugan Qingzhi Tablets during the Treatment of High-Fat-Diet-Induced Nonalcoholic Fatty Liver Disease.

BACKGROUND: Accumulative evidence showed that gut microbiota was important in regulating the development of nonalcoholic fatty liver disease (NAFLD). Hugan Qingzhi tablet (HQT), a lipid-lowering and anti-inflammatory medicinal formula, has been used to prevent and treat NAFLD. However, its mechanism of action is unknown. The aim of this study was to confirm whether HQT reversed the gut microbiota dysbiosis in NAFLD rats. METHODS: We established an NAFLD model of rats fed with a high-fat diet (HFD), which was given different interventions, and measured the level of liver biochemical indices and inflammatory factors. Liver tissues were stained with hematoxylin-eosin and oil red O. Changes in the gut microbiota composition were analyzed using 16S rRNA sequencing. RESULTS: The hepatic histology and biochemical data displayed that HQT exhibited protective effects on HFD-induced rats. Moreover, HQT also reduced the abundance of the Firmicutes/Bacteroidetes ratio in HFD-fed rats and modified the gut microbial species at the genus level, increasing the abundances of gut microbiota which were reported to have an effect on relieving NAFLD, such as Ruminococcaceae, Bacteroidales_S24-7_group, Bifidobacteria, Alistipes, and Anaeroplasma, and significantly inhibiting the relative abundance of Enterobacteriaceae, Streptococcus, Holdemanella, Allobaculum, and Blautia, which were reported to be potentially related to NAFLD. Spearman's correlation analysis found that [Ruminococcus]_gauvreauii_group, Lachnoclostridium, Blautia, Allobaculum, and Holdemanella exhibited significant (p < 0.001) positive correlations with triglyceride, cholesterol, low-density lipoprotein cholesterol, interleukin-6, interleukin-1ß, tumor necrosis factor-α, and body weight and negative correlations with high-density lipoprotein cholesterol (p < 0.001). The norank_f__Bacteroidales_S24-7_group and Alistipes showed an opposite trend. Moreover, the HQT could promote flavonoid biosynthesis compared with the HFD group. CONCLUSION: In summary, the HQT has potential applications in the prevention and treatment of NAFLD, which may be closely related to its modulatory effect on the gut microbiota.

Data for iTRAQ-based quantification of the effect of HuganQingzhi on non-alcoholic fatty liver disease in rats.

The data presented in this article are related to the research article entitled "Isobarictags for relative and absolute quantitation (iTRAQ) -based proteomics for the investigation of the effect of HuganQingzhi on non-alcoholic fatty liver disease in rats" (Yao et al., 2017) [1]. This article describes the effect of HuganQingzhi on non-alcoholic fatty liver disease in rats at the level of the proteome (HFD: control, HH: control, HH: HFD, respectively). The field dataset is available to criticize or extended analyzes in public.

Isobaric Tags for Relative and Absolute Quantitation (iTRAQ)-Based Proteomic Analysis of Hugan Qingzhi and Its Protective Properties against Free Fatty Acid-Induced L02 Hepatocyte Injury.

In previous research, Hugan Qingzhi, a traditional Chinese medicine, was shown to have protective effects against hepatic steatosis. However, its activity against non-alcoholic fatty liver disease (NAFLD) and the mechanisms by which it exerts its effects remain unknown. In the present study, the effects of Hugan Qingzhi on free fatty acid (FFA)-induced L02 cells were examined. The techniques of iTRAQ labeling, together with strong cation exchange-non-liquid chromatography-tandem mass spectrometry (SCX-non-LC-MS/MS) analysis and serum pharmacology, were used to evaluate the effects of Hugan Qingzhi-medicated serum on FFA-induced L02 hepatocyte injury. Results identified 355 differentially expressed proteins following FFA treatment, compared with a control group; 359 altered proteins in the Hugan Qingzhi high dose + FFA treatment group, compared with the FFA treatment group; and 365 altered proteins in the Hugan Qingzhi high dose + FFA treatment group, compared with the control group. Based on the Kyoto Encyclopedia of Gene and Genomes pathway enrichment analysis, it is concluded that several pathways including those of microbial metabolism in diverse environments, fatty acid metabolism, peroxisome proliferator activated receptor signaling, and mitogen-activated protein kinase signaling are closely associated with the effects of Hugan Qingzhi-medicated serum in FFA-induced L02 hepatocyte injury. Furthermore, several differentially expressed proteins, including heat shock protein 27 (HSP27), acetyl-CoA acetyltransferase 1, calnexin, and integrin-linked kinase, were validated by western blotting. A target-specific HSP27 siRNA was used to investigate further the function of HSP27, and it was found that HSP27 might have a key role in the observable effects of Hugan Qingzhi-medicated serum in FFA-induced L02 hepatocyte injury. The results not only confirmed that Hugan Qingzhi exhibits a significant protective effect in FFA-induced L02 hepatocyte injury, but also suggest insights into the mechanism of such protective effects.